Project Summary A major challenge in neuroscience is to comprehend how differentiating embryonic cells fashion de novo the working circuits and networks that make up a nervous system. We propose WormGUIDES, a resource that would facilitate understanding the cellular origins of an entire nervous system from all angles?cell birth, migration and differentiation; neurite formation, targeted outgrowth and bundling; and ultimately, synapse formation and tuning of functional circuits?with single cell resolution, from conception until hatching. Building on the innovations we established in the first funding cycle, we propose to use ShootingStar, a novel platform for real-time cell tracking and optical manipulation to label single neurons, and isotropic high-resolution diSPIM imaging to capture nerve bundling dynamics and single cell outgrowth in the early developing embryo when the major architecture of the nervous system is constructed. We will also assign all nuclear identities and positions from the first cell division until hatching. Finally, we will share the knowledge with the community through WormGUIDES Atlas software, displaying models, supporting images and primary data. Completion of the WormGUIDES resource will ultimately complement the existing cell lineage, adult nervous system structure, and collective genomic and genetic data for the worm, creating a fourth pillar of systems-level knowledge that will enhance our understanding of neurodevelopment.